iGEM week 2: 6/1 - 6/4
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Overview
Bacterial Decoder
Resistance
Precipitation
Cell Surface Engineering
Magnetite
Possible Metals:
• Mercury
• Arsenic
• Cadmium
• Nickel
• (Gold)
• Zinc
• (Silver)
What metals are
feasible to work with?
Bacterial Decoder
Constitutive Promoter
RBS
OmpA
GFP
Terminator
Plac
MicA
Ptet
MicA
Plac + Ptet
MicF
Ptet
RBS
OmpF
CFP
Terminator
Plac
RBS
OmpF
YFP
Terminator
Plac + Ptet
RBS
OmpA
RFP
Terminator
• Put both regulators and target on high copy
plasmids
• You have two GFPs? thanks
• Compare regulation to riboregulators
(BBa_J01008 BBa_J01010 BBa_J01080 BBa_J01086)
Tranformation/Precipitation of Heavy
Metals - Why
Heavy Metals are toxic to life, particularly in high concentrations
Disease Causing
Alleviate contamination of water supply
Help conserve habitable environment
We'll eventually run out of space to dig for storage
In terms of manufacturing, precipitation of these metals may
lead to bionanofabrication applications.
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quantum dots, single-electron transistors, fuel cells, fluorescent labelling, DNA/RNA detection,
biomedical diagnostic devices, biosensors, nanocomputers, drug and gene transport systems and
carbon nanotubes
Making bacteria resistant to these metals is the theoretical first
step towards a more efficient water cleaning system or
manufacturing system.
Cell Surface Engineering
Make a chimeric protein containing a metallothionein
Fuse cell membrane protein with metallothionein
Cell will display MTs on surface, and can collect soluble metals
that way
Applications:
Bioremediation of heavy metals in water/soil
Biomining
Problem: This seems too simple, we'd like to think of a way to
take it further than just simply collecting metals.... ideas??
Valls, M, Gonzalez-Duarte R, Atrian
S, Lorenzo V (1998)
Bioaccumulation of heavy metals
with protein fusions of
metallothionein to bacterial OMPs.
Biochimie 80: 855-861
Marc Valls, Sílvia Atrian, Víctor de
Lorenzo & Luis A. Fernández
Nature Biotechnology 18, 661 - 665
(2000)
Hg
• Resistance could be accomplished using: MerP, MerT, and
MerA (mercuric reductase)
• This process involves the reduction of Hg+2 to Hg0
• Export?
• 2ppb limit for
drinking water
• Could we work with
mercury?
Arsenic
The resistance pathway uses reduction however, it doesn't
involve reducing As to it's elemental form
Resistance Pathway:
ArsR: Regulatory Repressor
ArsB: Efflux Pump
ArsC: Arsenate Reductase
Alternatively, periplasmic
oxidase and reductase exist
Cadmium
Resistance is well defined, however the components involved
in reduction are less clear. May be related to resistance.
metals accumulate in the periplasm where they form metal
bicarbonates and carbonates that crystallize on cellular bound
metals.
alkaline pH in periplasm
due to protons being
pumped out
Nickel
• yohM
o codes for nickel and cobalt resistance.
o present in E.coli
o 825 nucleotides long
o stronger promotor should result in better
resistance.
• NreB and NrsD
o present in the microorganism R.
metallidurans
o could provide exclusive resistance to Ni.
Gold
Gold resistance is primarily conferred by 3 genes in Salmonella:
• golT, golS and golB
o golT - P-type ATPase efflux protein
o golS - Gold-dependent transcription factor for golTSB
o golB - Gold-binding protein
Gold reduction occurs by reducing Au(II) to Au(0)
• Several bacteria can naturally do this
• Pathway and proteins unknown for all of them
Expensive!
• $80/gram
Zinc
There is a zinc metallothionein gene ZmtA that we could possibly use
for cell surface engineering. It comes from Synechococcus elongatus
PCC 7942. It is a gram-negative bacteria. There is also a repressor for
this gene, ZmtB.
There is a zinc, cobalt, and lead efflux system called ZntA that is in
Escherichia coli str. K-12 substr. MG1655.
Lastly is a zinc-responsive transcriptional regulator ZntR Escherichia
coli str. K-12 substr. MG1655.
Microbial precipitation of zinc is done through complexing zinc with
sulfur, not reducing it to elemental zinc
• Mechanism unknown
Silver
Silver resistance has been identified : SilCBA operon: efflux
system
Present in E. coli and S. Typhimirium
Magnetite
• Fe(II)O + Fe(III)2O3
• Out ~25 proteins involved in magnetosome formation in
Magnetosprillum magneticum, 5 genes are involved in
magnetite production from iron: mms6, and mamGFDC.
• No intermediates between iron and magnetite
• Iron saturation is required
• 2 genes are involved in the uptake of iron: mamA, mamB
• oxygen conditions and pH determine the efficacy of
crystalization (optimal at low oxygen/anoxic conditions, high
hydrogen partial pressure, slightly reducing conditions)
• Is it possible to try and produce magnetite without the
magnetosome?
Questions